Size selection of RNA using poly(A) polymerase

ABSTRACT

This disclosure provides, among other things, a method for making a cDNA library. In some embodiments the method may comprise adding a polyA tail to the longer RNA fragments but not the shorter RNA fragments in a sample by incubating the population of RNA fragments with a polyA polymerase, wherein the reaction conditions used preferentially tail only the longer fragments but not the shorter fragments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/145,911, filed on Sep. 28, 2018, which application is incorporated byreference herein.

BACKGROUND

Certain cDNA library preparation methods involve fragmenting mRNA andthen reverse transcribing the resultant fragments of mRNA to make cDNA.In these methods, cleavage typically occurs at random or semi-randompositions and, as such, the population of RNA fragments made by suchmethods typically contains RNA fragments of different lengths, whereinat least some of the fragments are relatively small, i.e., less than 20nucleotides in length. These short fragments are problematic becausetheir cDNA copies are amplified very efficiently but their sequences(particularly for fragment that are 10-15 in length) are not alwaysuniquely mappable to a transcriptome. This problem can be potentiallyavoided by performing a physical size selection of the cDNAs or theamplification products. However, because of the imprecise nature ofphysical size selection methods it is impossible to eliminate cDNAcopies of the shorter RNAs without also eliminating cDNA copies of manyof longer RNAs, A better way for selecting RNA molecules by size istherefore needed.

SUMMARY

This disclosure provides, among other things, a method for processing anRNA sample. In some embodiments, the method may comprise: (a)fragmenting a sample comprising RNA (e.g., a sample that may comprise atleast mRNA, e.g., at least mRNA and lncRNA) to produce a population ofRNA fragments of different lengths, wherein the population of RNAfragments comprises longer RNA fragments and shorter RNA fragments, (b)incubating the population of RNA fragments with a polyA polymerase underconditions wherein the polyA polymerase preferentially adds a polyA tailto the longer RNA fragments and not the shorter fragments, to producetailed RNA; (c) hybridizing the tailed RNA to an oligo(dT)oligonucleotide; and (d) reverse transcribing the tailed RNA using theoligo(dT) oligonucleotide as a primer to produce a cDNA library orenriching for the tailed RNA by washing away RNA molecules that are nothybridized to the oligo(dT) oligonucleotide.

The present method is based in part on the discovery that polyApolymerase has a preference for longer RNA substrates. This discoverycan be applied in an in vitro polyadenylation reaction in order topolyadenylate only the longer RNA fragments and thereby eliminateshorter RNA fragments from future analysis. The preference of polyApolymerase for shorter substrates can be tuned by varying the reactionconditions. In many cases, limiting the activity of the enzyme in apolyadenylation reaction will result in the preferential polyadenylationof only the longer RNA fragments. This can be accomplished, for example,by reducing the amount of enzyme in the reaction, by reducing the amountof free Mg²⁺ in the reaction, or by modifying other reaction conditions.The reaction conditions can be tuned so that RNA fragments that are atleast 20 ribonucleotides in length are preferentially polyadenylatedrelative to the smaller RNA fragments that are 15 nucleotides orshorter.

As will be explained in greater detail below, the method may findparticular use in analyzing samples that contain both “long” RNAs (suchas mRNA, lncRNA, etc.) and small RNAs. After fragmentation, the RNAfragments may have a median size of at least 20 nucleotides, the smallRNAs may have a median size in the range of 20 to 40 nucleotides and theshorter, undesirable, RNA fragments may be 15 nucleotides or shorter.The preference of polyA polymerase for longer substrates can be used topreferentially polyadenylate both the small RNAs and the longer RNAfragments, but not the smaller RNA fragments. The method may be employedon any RNA sample that comprises RNAs that are at least 40 nucleotidesin length. The RNA molecules in such samples may have a median size ofat least 40 nucleotides or at least 100 nucleotides, for example.

The present method, because it is capable of discriminating between RNAmolecules that differ in length by only 5-10 nucleotides, is believed toprovide higher resolution than other methods that physically removesmaller RNA fragments from the sample (e.g., by size exclusionchromatography and/or bead-based size selection).

BRIEF DESCRIPTION OF THE FIGURES

Some aspects of the present invention may be best understood from thefollowing detailed description when read in conjunction with theaccompanying drawings. It is emphasized that, according to commonpractice, the various features of the drawings are not to scale. Indeed,the dimensions of the various features are arbitrarily expanded orreduced for clarity. Included in the drawings are the following figures.

FIG. 1 schematically illustrates some of the principles of an embodimentof the present method.

FIG. 2 schematically illustrates how fragmentation can be accomplishedby RNAseH treatment of a DNA/RNA hybrid.

FIG. 3 schematically illustrates how shorter RNA fragments can beexcluded from a cDNA library using the present method.

FIG. 4 is a bar graph showing data from the first experiment, asdescribed in the examples.

FIG. 5 is a bar graph showing data from the second experiment, asdescribed in the examples.

DEFINITIONS

Before describing exemplary embodiments in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used in the description.

Numeric ranges are inclusive of the numbers defining the range. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; and, amino acid sequences are written left to right inamino to carboxy orientation, respectively.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. For example, the term “a primer”refers to one or more primers, i.e., a single primer and multipleprimers. It is further noted that the claims can be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

The term “RNA sample”, as used herein, relates to a mixture ofmaterials, typically, although not necessarily, in liquid form, e.g., Inthe form of an aqueous solution, containing one or more RNA molecules.An RNA sample may be obtained from cells, e.g., mammalian cells, forexample. An RNA sample may contain any number of distinguishable RNAmolecules. For example, in some embodiments, an RNA sample may contain apopulation of different RNA molecules, in which case it may contain morethan 1,000, more than 10,000, more than 50,000, or more than 100,000 upto 1M or more different species of RNA, i.e., RNA molecules of differentsequence. An RNA sample may contain long RNA molecules such as mRNAmolecules, which are typically at least 100 nt in length (e.g., 200 ntto 10 kb in length) and have a median length in the range of 500-5,000nt, as well as, for example, long intergenic noncoding RNAs (lincRNAs).An RNA sample may additionally contain a variety of small non-codingregulatory RNAs that may be generically referred herein to as “smallRNAs”, e.g., microRNAs, tiny non-coding RNAs, piwi-interacting smallRNAs (piRNAs), small modulatory RNAs, and snoRNAs, etc. Small RNAs aretypically below 100 nt in length and have a median length in the rangeof 20 nt to 40 nt. An RNA sample may additionally contain rRNAmolecules, tRNA molecules, pre-miRNA molecules, and long non-coding RNAmolecules such as large intergenic RNA (lincRNA) molecules. Unlessotherwise indicated, an “RNA sample” may have any type ofnaturally-occurring RNA, including those described above and potentiallyothers.

The term “nucleotide” is intended to include those moieties that containnot only the known purine and pyrimidine bases, but also otherheterocyclic bases that have been modified. Such modifications includemethylated purines or pyrimidines, acylated purines or pyrimidines,alkylated riboses or other heterocycles. In addition, the term“nucleotide” includes those moieties that contain hapten or fluorescentlabels and may contain not only conventional ribose and deoxyribosesugars, but other sugars as well. Modified nucleosides or nucleotidesalso include modifications on the sugar moiety, e.g., wherein one ormore of the hydroxyl groups are replaced with halogen atoms or aliphaticgroups, are functionalized as ethers, amines, or the likes. Nucleotidesmay include those that when incorporated into an extending strand of anucleic acid enables continued extension (non-chain terminatingnucleotides) and those that prevent subsequent extension (e.g. chainterminators).

The term “nucleic acid” and “polynucleotide” are used interchangeablyherein to describe a polymer of any length, e.g., greater than about 2bases, greater than about 10 bases, greater than about 100 bases,greater than about 500 bases, greater than 1000 bases, up to about10,000 or more bases composed of nucleotides, e.g., deoxyribonucleotidesor ribonucleotides, and may be produced enzymatically or synthetically(e.g., PNA as described in U.S. Pat. No. 5,948,902 and the referencescited therein) which can hybridize with naturally occurring nucleicacids in a sequence specific manner analogous to that of two naturallyoccurring nucleic acids, e.g., can participate in Watson-Crick basepairing interactions. Naturally occurring nucleotides include guanine,cytosine, adenine, thymine and uracil (G, C, A, T, and U).

The terms “ribonucleic acid” and “RNA” as used herein mean a polymercomposed of ribonucleotides.

The terms “deoxyribonucleic acid” and “DNA” as used herein mean apolymer composed of deoxyribonucleotides.

“Isolated” or “purified” generally refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptidecomposition) such that the substance comprises a significant percent(e.g., greater than 1%, greater than 2%, greater than 5%, greater than10%, greater than 20%, greater than 50%, or more, usually up to about90%-100%) of the sample in which it resides. In some cases, an isolatedsubstance may be dissolved in a liquid, e.g., an aqueous liquid. Incertain embodiments, a substantially purified component comprises atleast 50%, 80%-85%, or 90-95% of the sample. Techniques for purifyingpolynucleotides and polypeptides of interest are well-known in the artand include, for example, ion-exchange chromatography, affinitychromatography, sedimentation according to density, precipitation,solvent extraction and solid phase purification using a column or beads.Generally, a substance is purified when it exists in a sample in anamount, relative to other components of the sample, that is not foundnaturally.

The term “oligonucleotide”, as used herein, denotes a single-strandedmultimer of nucleotides from about 2 to 500 nucleotides, e.g., 2 to 200nucleotides. Oligonucleotides may be synthetic or may be madeenzymatically, and, in some embodiments, are 4 to 50 nucleotides inlength. Oligonucleotides may contain ribonucleotide monomers (i.e., maybe RNA oligonucleotides) or deoxyribonucleotide monomers.Oligonucleotides may be 5 to 20, 11 to 30, 31 to 40, 41 to 50, 51 to 60,61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200, up to 500nucleotides in length, for example. The term “duplex”, “hybrid” or“double-stranded” as used herein refers to nucleic acids that have twostrands that are bound together by based pairing.

The term “complementary” as used herein refers to a nucleotide sequencethat base-pairs by non-covalent bonds to a target nucleic acid ofinterest. In the canonical Watson-Crick base pairing, adenine (A) formsa base pair with thymine (T), as does guanine (G) with cytosine (C) inDNA. In RNA, thymine is replaced by uracil (U). As such, A iscomplementary to T and G is complementary to C. In RNA, A iscomplementary to U and vice versa. Typically, “complementary” refers toa nucleotide sequence that is at least partially complementary. The term“complementary” may also encompass duplexes that are fully complementarysuch that every nucleotide in one strand is complementary to everynucleotide in the other strand in corresponding positions. In certaincases, a nucleotide sequence may be partially complementary to a target,in which not all nucleotides are complementary to every nucleotide inthe target nucleic acid in all the corresponding positions.

The terms “determining”, “measuring”, “evaluating”, “assessing”,“analyzing”, and “assaying” are used interchangeably herein to refer toany form of measurement or analysis, and include determining if anelement is present or not. These terms include both quantitative and/orqualitative determinations. Assessing may be relative or absolute.“Assessing the presence of” includes determining the amount of somethingpresent, as well as determining whether it is present or absent.

As used herein, the term “total cellular RNA” is an RNA sample thatcontains at least tRNA, rRNA, mRNA, lincRNA and small RNA.

As used herein, the term “depleted”, in the context of a total cellularRNA sample that has been depleted for tRNA, rRNA, or another type ofRNA, is total cellular RNA sample from which tRNA, rRNA, or another typeof RNA has been subtracted, i.e., removed, degraded or substantiallyreduced.

As used herein, the term “adaptor” refers to an oligonucleotide that maybe composed of any type of nucleotide. An adaptor may be, e.g., an RNAadaptor, a DNA adaptor, or it may be composed of both ribonucleotidesand deoxyribonucleotides or analogs thereof. An adaptor may be of 5-50bases, e.g., 10 to 30 bases, in length or longer depending on theapplication. An adaptor may contain a molecular barcode, restrictionsites and/or primer binding sites, depending on the application. In themethods described below, at least the 3′ end of the adaptor can be RNA.In some embodiments, an adaptor can contain a molecular barcode (e.g.,an “index” or “indexing” sequence).

As used herein, the terms “3′-OH” and “3′-hydroxyl” refer to anucleotide at the 3′ terminus of a nucleic acid, where the nucleotidehas a hydroxyl group at the 3′ position.

As used herein, the term “5′-P” or “5′-phosphate” refers to a nucleotideat the 5′ terminus of a nucleic acid, where the nucleotide has aphosphate group at the 5′ position.

As used herein, the term “cDNA library” refers to a collection of DNAs,or library, synthesized from a template RNA and are thereforecomplimentary to the template RNA. The cDNA library can be sequenced,labeled, amplified and/or cloned, depending on how it is going to beused.

As used herein, the term “RNA:cDNA hybrid” refers to a product afterfirst-strand cDNA synthesis catalyzed by reverse transcriptase using RNAas a template. An “RNA-cDNA hybrid” can be full-length if the cDNAportion includes the complete sequence of the 5′-ends of the templatemRNA. RNA:DNA hybrids can also be made by hybridizing DNAoligonucleotides (which can be sequence-specific or random) with RNA.

As used herein, the term “template” refers to the substrate RNA for thereverse transcriptase to make cDNA. The template RNA is the target in amixed population of RNA molecules for enrichment.

The term “non-naturally occurring” refers to a composition that does notexist in nature. Any protein described herein may be non-naturallyoccurring, where the term “non-naturally occurring” refers to a proteinthat has an amino acid sequence and/or a post-translational modificationpattern that is different to the protein in its natural state. Forexample, a non-naturally occurring protein may have one or more aminoacid substitutions, deletions or insertions at the N-terminus, theC-terminus and/or between the N- and C-termini of the protein. A“non-naturally occurring” protein may have an amino acid sequence thatis different to a naturally occurring amino acid sequence (i.e., havingless than 100% sequence identity to the amino acid sequence of anaturally occurring protein) but that is at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, at least 98% or at least 99%identical to the naturally occurring amino acid sequence. In certaincases, a non-naturally occurring protein may contain an N-terminalmethionine or may lack one or more post-translational modifications(e.g., glycosylation, phosphorylation, etc.) if it is produced by adifferent (e.g., bacterial) cell. A “mutant” or “variant” protein mayhave one or more amino acid substitutions relative to a wild-typeprotein and may include a “fusion” protein. The term “fusion protein”refers to a protein composed of a plurality of polypeptide componentsthat are unjoined in their native state. Fusion proteins may be acombination of two, three or even four or more different proteins. Theterm polypeptide includes fusion proteins, including, but not limitedto, a fusion of two or more heterologous amino acid sequences, a fusionof a polypeptide with: a heterologous targeting sequence, a linker, anepitope tag, a detectable fusion partner, such as a fluorescent protein,β-galactosidase, luciferase, etc., and the like. A fusion protein mayhave one or more heterologous domains added to the N-terminus,C-terminus, and or the middle portion of the protein. If two parts of afusion protein are “heterologous”, they are not part of the same proteinin its natural state. In the context of a nucleic acid, the term“non-naturally occurring” refers to a nucleic acid that contains: a) asequence of nucleotides that is different to a nucleic acid in itsnatural state (i.e. having less than 100% sequence identity to anaturally occurring nucleic acid sequence), b) one or more non-naturallyoccurring nucleotide monomers (which may result in a non-naturalbackbone or sugar that is not G, A, T or C) and/or c) may contain one ormore other modifications (e.g., an added label or other moiety) to the5′-end, the 3′ end, and/or between the 5′- and 3′-ends of the nucleicacid.

In the context of a composition, the term “non-naturally occurring”refers to: a) a combination of components that are not combined bynature, e.g., because they are at different locations, in differentcells or different cell compartments; b) a combination of componentsthat have relative concentrations that are not found in nature; c) acombination that lacks something that is usually associated with one ofthe components in nature; d) a combination that is in a form that is notfound in nature, e.g., dried, freeze dried, crystalline, aqueous; and/ore) a combination that contains a component that is not found in nature.For example, a preparation may contain a “non-naturally occurring”buffering agent (e.g., Tris, HEPES, TAPS, MOPS, tricine or MES), adetergent, a dye, a reaction enhancer or inhibitor, an oxidizing agent,a reducing agent, a solvent or a preservative that is not found innature.

The term “primer” refers to an oligonucleotide, either natural orsynthetic, that is capable, upon forming a duplex with a polynucleotidetemplate, of acting as a point of initiation of nucleic acid synthesisand being extended from its 3′ end along the template so that anextended duplex is formed. The sequence of nucleotides added during theextension process is determined by the sequence of the templatepolynucleotide. Usually primers are extended by a DNA polymerase or areverse transcriptase. Primers are generally of a length compatible withtheir use in synthesis of primer extension products, and are usually arein the range of between 6 to 100 nucleotides in length, such as 10 to75, 15 to 60, 15 to 40, 18 to 30, 20 to 40, 21 to 50, 22 to 45, 25 to40, and so on, more typically in the range of between 18-40, 20-35,21-30 nucleotides long, and any length between the stated ranges.Primers are usually single-stranded. Primers have a 3′ hydroxyl.

The term “oligo-dT primer” refers to a primer that is capable of primingcDNA synthesis from a polyA tail. With the possible exception of thelast one or two nucleotides, an oligo-dT primer may have a 3′ endsequence that has a string of thymines. Such a primer may have a 5′tail, as shown in FIG. 3. In some embodiments, oligo-dT primer may beanchored and may have the following sequence: TTTTTTTTTTTTTTTVN (SEQ IDNO:1), where V is G, A or C or analog thereof and N is any nucleotide oranalog thereof. The T in such an oligonucleotide may be a T analog inthat it is capable of specifically base pairing with an A.

The term “sequence-specific primer” for the purpose of reversetranscribing an RNA is intended to refer to a primer that hybridizes toa unique sequence in mRNA or a target RNA. Sequence-specific primers donot have a random sequence and are not made of a single nucleotide.Random primers and oligo(T) primers are not sequence specific primers.

The term “cDNA copy” refers to a DNA molecule that has the reversecomplement of an RNA molecule (i.e., first strand cDNA) or a DNAmolecule that has the same sequence as an RNA molecule except that theUs are T's (i.e., second strand cDNA). The RNA molecule can by any typeof RNA, e.g., a small RNA, a fragment or mRNA, or a fragment of lncRNA,etc.

The term “reaction conditions” refers to the temperature of a reaction,the length of time the reaction is incubated for, and/or the componentsof the reaction (e.g., the amounts of salt, enzyme, pH, divalent cationused, enzyme inhibitors, etc.).

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION

Before the various embodiments are described, it is to be understoodthat the teachings of this disclosure are not limited to the particularembodiments described, and as such can, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present teachings will be limited onlyby the appended claims.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way. While the present teachings are described in conjunction withvarious embodiments, it is not intended that the present teachings belimited to such embodiments. On the contrary, the present teachingsencompass various alternatives, modifications, and equivalents, as willbe appreciated by those of skill in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, some exemplarymethods and materials are now described.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentclaims are not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided can be differentfrom the actual publication dates which can need to be independentlyconfirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentteachings. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

FIG. 1 illustrates some of the principles of an embodiment of themethod. With reference to FIG. 1, some embodiments of the method maycomprise fragmenting a sample comprising mRNA, e.g., mRNA 2 to produce apopulation of RNA fragments 4 of different lengths, wherein thepopulation of RNA fragments 4 comprises longer RNA fragments 6 andshorter RNA fragments 8. This fragmentation step may be done in avariety of different ways. For example (and as will be described ingreater detail below), the RNA sample 2 may contain cDNA:mRNA hybrids(i.e., may comprise reverse transcription products primed using, e.g.,an oligo(dT) primer), and the fragmenting of the mRNA may be done bytreating the sample with RNAseH (see U.S. application Ser. No.15/818,469, filed on Nov. 20, 2017, which is incorporated by referenceherein). Fragmentation can also be done by hybridizing DNAoligonucleotides (which may be sequence-specific or random, for example)to the RNA to produce DNA:mRNA hybrids and then treating the hybridswith RNAseH. Alternatively, the mRNA may be fragmented by heating themRNA (e.g., to a temperature of at least 60° C.) for a period of atleast 5 minutes in the presence of a divalent cation (e.g., Mg²⁺ orZn²⁺)) or another method (see, for example, Leven et al Nat Methods.2010 7: 709-715). Depending on the fragmentation conditions used, thefragmentation step can result in longer fragments are at least 20ribonucleotides in length and the shorter fragments 15 or lessribonucleotides in length. After the fragments have been made, then thelonger fragments 6 are selectively adenylated using polyA polymerase toproduce tailed RNA 9. Preferentially adding a polyA tail to the longerRNA fragments but not the shorter RNA fragments is done by incubatingthe population of RNA fragments 4 with a polyA polymerase underconditions wherein the polyA polymerase preferentially adds a polyA tailto the longer RNA fragments and not the shorter fragments, to producetailed RNA 9. Suitable reaction conditions limit the activity of thepolyA polymerase in the reaction. The activity that is effective forpreferentially tailing only the longer fragments is generally much lessthan (e.g., at least 1/10^(th) of) the activity that typically used in apolyadenylation reaction. Under these conditions, the reaction is notbelieved to go to completion and the limited enzyme activity selects forthe longer substrates. The activity of the enzyme can be limited in avariety of different ways. For example, in the present method thereaction conditions (i.e., the polyadenylation reaction buffer) maycomprise polyA polymerase at a concentration of less than 0.02 U/uL(e.g., at a concentration of 0.002 U/uL to 0.02 U/uL or 0.003 U/uL to0.015 U/uL of polyA), where unit of polyA polymerase is defined as theamount of enzyme that will incorporate 1 nmol of ATP into acid-insolublematerial in 10 minutes at 37° C., in a reaction that contains theenzyme, a 15-mer RNA oligonucleotodie, 1× reaction buffer (50 mMTris-HCl, 250 mM NaCl, 10 mM MgCl2, pH 7.9 @ 25° C.), 1 mM ATP, 2.5 mMMnCl2 and 3H-ATP, analyzed using the method of Sambrook and Russell(Molecular Cloning, v3, 2001, pp. A8.25-A8.26). Typical polyadenylationreactions use polyA polymerase at a concentration of 0.333 U/uL to 0.5U/uL and, as such, the present reaction may use less than 1/20^(th) ofthe concentration of polyA polymerase of a conventional reaction. Forexample, a 10 uL to 20 uL polyadenylation reaction may comprise lessthan 0.25 units of the polyA polymerase (e.g., 0.05 to 0.15 units ofpolyA polymerase). Typical polyadenylation reactions use 2-5 units ofpolyA polymerase. Alternatively, the activity of the polyA polymerasemay be limited using a reduced amount of divalent cation in the reactionbuffer. PolyA polymerase requires a divalent cation (typically Mg²⁺) foractivity and its activity can be attenuated in a reaction that has adecreased amount of divalent cation (e.g., Mg²⁺). As such, in someembodiments, the reaction conditions (i.e., the polyadenylation reactionbuffer) may comprise 0.6 mM to 1.2 mM (0.75 mM to 1.0 mM) of a divalentcation, e.g., Mg²⁺, where a typical polyA tailing reaction may containabout 10 mM Mg²⁺ (see the reaction conditions for determining a unit ofpolyA polymerase above). Typical reactions include an incubation for atime period of under 1 hour (e.g., 5 mins to 30 mins, up to 1 hr). Thesame effect can likely be created by altering other reaction conditions,e.g., the time or temperature of the incubation, or by manipulatingother components of the reaction. A typical polyadenylation reaction mayhave 1 ng to 1 ug of fragmented RNA, but this may vary. For example, ifone uses 1 ng of total RNA as an input into a fragmentation reactionthat is targeted to polyA+ RNA, only a small fraction of the RNA, e.g.,as low as 10 pg, may be fragmented.

After the tailed RNA 9 has been produced, the tailed RNA may behybridized with an oligo(dT) oligonucleotide. After hybridization to theoligo(dT) oligonucleotide, the method may comprise reverse transcribingthe tailed RNA using the oligo(dT) oligonucleotide as a primer toproduce a cDNA library, or enriching for the tailed RNA by washing awayRNA molecules that are not hybridized to the oligo(dT) oligonucleotide.In the latter embodiment, the tailed RNA 9 can be affinity selectedusing an oligo(dT) column, if desired. As shown in FIG. 1, after thetailed RNA 9 has been produced, the tailed RNA 9 may be reversetranscribed to produce a cDNA library 10. In some embodiments and asshown in FIG. 1, the tailed RNA 9 may be reverse transcribed using anoligo(dT) primer (e.g., an anchored oligo(dT) primer that may have a 5′tail). As illustrated in FIG. 1, only the polyadenylated RNAs (i.e., thepolyadenylated RNAs but not the unpolyadenlyated RNAs) will be copiedinto cDNA because the oligo(dT) primer will only hybridize to thepolyadenylated RNAs. The method shown in FIG. 1 therefore provides a wayto preferentially make cDNA from longer RNAs. As shown, the method maybe done without in the absence of a physical size selection step thatinvolves incubating the RNA fragments 4 with a matrix that selectivelybinds to either the shorter RNA fragments or longer RNA fragments.However, a physical size selection step can be employed, if desired.

FIG. 2 illustrates an example of how fragments of RNA can be producedusing RNaseH. It should be noted that the method shown in this figure isnot the only method that can be used to make fragments of RNA. Themethod illustrated in FIG. 2 may comprise reverse transcribing RNAsample 12 that comprises polyA tailed mRNA to produce first strand cDNAproduct 14. The polyA tailed mRNA may be from a cell (e.g., a eukaryoticcell) or made by adding a polyA tail to a population of mRNAs that arenot polyA tailed using polyA polymerase. As shown, the first strand cDNAproduct 14 comprises cDNA:RNA hybrids that comprise an RNA and a cDNAcopy of the RNA. In the embodiment shown in FIG. 2, the reversetranscription may be primed using an oligo(dT) primer (e.g., an anchoredoligo(dT) primer). In other embodiments, the initial reversetranscription step may be done using one more sequence-specific primers(e.g., primers that hybridize to unique sequences in the RNA) or randomprimers. Alternatively, the DNA:RNA hybrids may be made by hybridizingsequence-specific or random oligonucleotides to an RNA sample. The RNAsample may contain, for example, total cellular RNA, total RNA that hasbeen depleted for one or more types of RNA (e.g., rRNA and/or tRNA),lncRNA, size-selected RNA or mRNA and small RNA, for example, althoughother combinations are contemplated. In some embodiments, the reversetranscription may be done by a “hot start” procedure in which twocomplementary mixtures are pre-heated to the incubation temperatureprior to mixing them together. In these embodiments, the initial reversetranscription step may be done by: (i) pre-heating a first mixturecomprising the primer and the RNA sample to a temperature in the rangeof 40-80 degrees, (ii) pre-heating a second mixture comprising thereverse transcriptase to a temperature in the range of 40-80 degrees,(iii) admixing the first and second mixtures to produce a reaction mix;and incubating the reaction mix at a temperature of 40-80 degrees for asufficient time (e.g., at least 5 minutes), to produce the first strandcDNA product. As shown in FIG. 2, the method may comprise treating thefirst stand cDNA product 14 with RNAseH to produce a digested sample 16that comprises fragments of the mRNA. In practice, the number fragmentsof mRNA per DNA:mRNA hybrid may vary greatly based on the length of theDNA:mRNA hybrids and the number of cleavage events that occur perDNA:mRNA hybrid (i.e., the number of times the RNAseH nicks the mRNAmolecule of the DNA:mRNA hybrid). In some embodiments, at least 90% ofthe DNA:mRNA hybrids may give rise to approximately 4 to 200 DNA:mRNAhybrids each. The median length of fragments 14 may be at least 20nucleotides (e.g., in the range of 20 to 100 or 20 to 50 nucleotides).As noted, the RNA may be fragmented using another method. Some of thealternative methods produce RNA fragments that have 5′ hydroxyl and a 3′phosphate and, as such, may require a phosphatase and/or kinasetreatment before moving on to the next step. mRNAs may be fragmented toa median length of between 10 to 200 nucleotides. In some embodiments,the median length of the fragments is 10-25 nucleotides. In otherembodiments the median length may be 40-150 nucleotides. In eitherembodiment, there will be a substantial number of molecules that are 15nucleotides or below in length, which can be eliminated using thepresent method.

In some embodiments, the reverse transcriptase and RNAseH activitiesrequired for the initial steps of the method are provided by differentenzymes. In these embodiments, the reverse transcription step may bedone using an RNAaseH⁻ reverse transcriptase, and the RNAseH treatmentmay be done using a separate enzyme. In some embodiments, the reversetranscription and/or the RNAseH may thermostable. In some embodiments,the initial reverse transcription step may be done at temperature in therange of 40° C. to 80° C. In some embodiments, the RNAseH treatment stepmay be done at a temperature in the range of 40° C. to 80° C., e.g., ata temperature in the range of 60° C. to 80° C. It is thought that at anelevated temperature incompletely digested mRNA fragments (e.g.,fragments that have a median length in the range of 15 to 50nucleotides) produced by RNAseH cleavage start to become disassociatedfrom the cDNA to which they were bound. RNAseH requires adouble-stranded substrate, and because their disassociation prevents thefragments from being a substrate for the RNAseH, mRNA fragments in therange of 15 to 50 nucleotides should not be digested any further, evenin an extended incubation. Thus, in some embodiments, the reactionconditions (the salt concentration and temperature) can be adjusted toproduce fragments of a pre-determined range of sizes. Higher salt and/ora higher incubation temperature should, in theory, result in apopulation of fragments that have a longer median length and lower saltand/or a lower incubation temperature should, in theory, result in apopulation of fragments that have a shorter median length. The length ofthe fragments can also be tailored by modifying the amount of enzymeused and/or the incubation conditions and/or by altering total RNA inputconcentration. In some embodiments, in order to avoid complete digestionof the mRNAs and to obtain mRNA fragments of the desired length, theamount of RNAseH used in the RNAseH treatment may be less than a tenthof the amount of RNAseH used for other reactions. For example, if 5units of RNAseH are typically used to digest mRNA to completion, then0.1 to 0.5 units of RNAseH (e.g., an amount in the range of 0.3 to 0.16units) may be used in the present method, where one unit of RNaseH isthe amount of enzyme which produces 1 nmol acid soluble ribonucleotidesfrom [3H]poly(A)×poly(dT) in 20 minutes at 37° C. under the conditionsused, using the method of Hillenbrand and Saudenbauer (Nucleic AcidsRes. 1982 10:833).

Next and as shown in FIG. 2, the method may comprise polyadenylating themRNA fragments to produce a cDNA library 18, using the method describedin FIG. 1.

The polyadenylated fragments produced by the method shown in FIGS. 1 and2 may be reverse transcribed using any suitable method. FIG. 3 shows oneexample of such a method although, as would be apparent, other methodsmay be used. For example, the 5′ adaptor sequence may be added to thereverse transcription product by template switching, rather thanligation. In the example shown in FIG. 3, the method may comprise addinga poly(A) tail onto the 3′ end of the longer RNA fragments 20 but notthe shorter RNA fragments 22 of a sample 24 using an polyA polymerase,as described above, to produce tailed RNA 26. As shown, the longerfragments 20 have an A tail whereas the shorter fragments 22 do not havean A tail in tailed RNA 26. Next, the fragments may be reversetranscribed by ligating a 5′ adaptor 28 to the tailed fragments (and theuntailed fragments), to produce an adaptor-tagged sample 30. In someembodiments the 5′ adaptor may be a single-stranded oligonucleotide inthe range of 5 to 20 nt in length (e.g., 6, 7, 8, 9, 10, 11 or 12 nt inlength), although adaptors having a length outside of this range mayalso be employed. The adaptor may be an RNA oligonucleotide, a DNAoligonucleotide or an oligonucleotide that comprises DNA and RNA. Theadaptor may be ligated onto the RNA molecules of the digested sampleusing an RNA ligase, e.g., T4 RNA ligase, using any of the methodsoutlined in Wang et al (RNA 2007 13: 151-159) or Lockhart et al (U.S.Pat. No. 6,344,316) among many others. The RNA ligase used in the methodcan be any suitable ligase. In some embodiments, T4 RNA ligase can beused, although a variety of other RNA ligases that have a preference forsingle-stranded substrates can be used instead. In some embodiments, theRNA ligase used may be thermostable. In these embodiments, the ligationreaction may be done at an elevated temperature that may be in the rangeof 40 to 80° C.

The longer fragments in the A-tailed, adaptor ligated sample 30 may bereverse transcribed using an oligo(dT) primer 32 (e.g., an anchoredoligo(dT) primer) to produce cDNA library 36. In the embodiment shown,oligo(dT) primer 32 contains an optional 5′ tail 34, which does nothybridize to the A-tailed, adaptor ligated RNAs 30. As shown in FIG. 3,the cDNA library 36 may be optionally amplified by PCR to produce anamplification product 40. This step may be done using a first primer 38that has a 3′ end that is the same as a sequence in the 5′ adaptor and asecond primer 39 that has a 3′ end that is the same as a sequence in thetail of the oligo(dT) primer. As shown, the amplification product 40contains amplicons of the longer RNA fragments but not the shorter RNAfragments. In some embodiments, the amplification product may besubjected to a size selection step to remove unincorporated primersand/or unwanted species such as rRNA fragments or snoRNAs prior toanalysis.

In some embodiments that use RNAseH to fragment the RNA, the cDNA thatis made in the first step of the method is not itself analyzed and, assuch, the cDNA molecules made in the initial step of the method may bedegraded (e.g., using a DNAse treatment), discarded (e.g., purified awayfrom the cDNA in the library by size separation) and/or diluted out (bypreferentially amplifying the cDNA molecules in the cDNA library) priorto sequencing.

In some embodiments, the initial sample (prior to fragmentation) maycomprise small RNAs that are in the range 20 and 50 nucleotides inlength and have a median length in the range of 20 nt to 40 nt, inaddition to mRNA. Small RNAs include microRNA (miRNA) molecules, tinynon-coding RNA (tncRNA) molecules and small modulatory RNA (smRNA)molecules, as well as others. In these embodiments, the initialfragmentation step may avoid fragmenting the small RNAs. For example, ifthe initial RNA is fragmented by heat in the presence of a divalentcation (which primarily cleaves longer RNAs because they contain morecleavage sites than short RNAs), then the cleavage reaction can beterminated before the small RNAs are significantly fragmented. In othercases, cleavage of the small RNAs can be avoided because they do notcontain a polyA tail. Specifically, if the fragmenting is done using themethod illustrated in FIG. 2, then the small RNAs should not befragmented because they do not have a polyA tail and will not be copiedinto cDNA in the initial reverse transcription step. RNAseH requires anDNA:RNA hybrid and, as such, only the RNAs that have been reversetranscribed (and not the small RNAs or other types of RNA that have notbeen reverse transcribed) should be cleaved by the RNAseH. If small RNAsare present in the sample, then the RNAs may be fragmented to a medianlength that is similar to the length of the small RNAs. Small RNAs canbe in the range of 20-29 nucleotides in length, and many small RNAs areapproximately 20-25 nucleotides in length. As such, if small RNAs andlonger RNAs (e.g., mRNAs and lncRNAs) are going to be analyzed, then theRNAs may be fragmented to a median length of between 10 to 100nucleotides, e.g., 15 to 50 nucleotides. As such, in some embodiments,the method may comprise fragmenting the sample to produce a populationof RNA fragments of different lengths, wherein the population of RNAfragments comprises: i. longer RNAs comprising unfragmented small RNAsand fragments of the mRNA and ii, shorter RNAs comprising fragments ofthe mRNA, adding a polyA tail to the longer RNAs but not the shorterRNAs by incubating the population of RNA fragments with a polyApolymerase under suitable reaction conditions (as described above), toproduce tailed RNA that contains tailed RNA fragments and tailedunfragmented small RNAs; (c) hybridizing the tailed RNA to an oligo(dT)oligonucleotide; and (d) reverse transcribing the tailed RNA using theoligo(dT) oligonucleotide as a primer, to produce a cDNA library orenriching for the tailed RNA by washing away RNA molecules that are nothybridized to the oligo(dT) oligonucleotide. For example, in someembodiments, the method may comprise reverse transcribing the tailed RNAto produce a cDNA library that comprises: i. copies of the small RNAsand ii. copies of the longer RNA fragments. In these embodiments, boththe small RNAs and the RNA fragments have a 5′ phosphate and a 3′hydroxyl and, as such, can be polyadenylated, ligated to adaptors, andreverse transcribed together in the same reaction (e.g., bypolyadenlyating the 3′ end of any longer RNAs using poly A polymerase,as discussed above, ligating a 5′ adaptor to any RNA molecules that havea 5′ phosphate, and reverse transcribing any molecules that have beentailed using an oligo(dT) primer, for example). The cDNA library maycontain the first strand cDNA made in the initial step of the method, ornot. In some cases, the first strand cDNA made in the initial step ofthe method may have been removed or degraded prior to making the cDNAlibrary. This embodiment of the method provides a way to analyze smallRNAs and mRNA in the same workflow.

In embodiments in which the cDNAs are sequenced, the cDNA library may beamplified using one or more primers that hybridize to the addedsequences (or their complements), as described above. In someembodiments, the primers used may have sequences that are compatiblewith the sequencing platform being used (e.g., P5 and P7 sequences,which sequences are compatible with Illumina's sequencing platform) andthe amplification products will have those sequences at their ends(e.g., P5 sequence at one and the P7 sequence at the other, if theIllumina sequencing platform is being used).

The sequencing step may be done using any convenient next generationsequencing method and may result in at least 10,000, at least 50,000, atleast 100,000, at least 500,000, at least 1M, at least 10M, at least100M, 1B or at least 10B sequence reads. In some cases, the reads arepaired-end reads. As would be apparent, the primers used foramplification may be compatible with use in any next generationsequencing platform in which primer extension is used, e.g., Illumina'sreversible terminator method, Roche's pyrosequencing method (454), LifeTechnologies' sequencing by ligation (the SOLiD platform), LifeTechnologies' Ion Torrent platform or Pacific Biosciences' fluorescentbase-cleavage method. Examples of such methods are described in thefollowing references: Margulies et al (Nature 2005 437: 376-80); Ronaghiet al (Analytical Biochemistry 1996 242: 84-9); Shendure (Science 2005309: 1728); Imelfort et al (Brief Bioinform. 2009 10:609-18); Fox et al(Methods Mol Biol. 2009; 553:79-108); Appleby et al (Methods Mol Biol.2009; 513:19-39) English (PLoS One. 2012 7: e47768) and Morozova(Genomics. 2008 92:255-64), which are incorporated by reference for thegeneral descriptions of the methods and the particular steps of themethods, including all starting products, reagents, and final productsfor each of the steps.

In certain embodiments, the cDNA that is sequenced may comprise a poolof cDNA libraries made from a plurality of different RNA samples,wherein the different cDNA libraries have a molecular barcode (in theadaptor or PCR primers) to indicate their source. In some embodimentsthe cDNAs being analyzed may be derived from a single source (e.g., asingle organism, virus, tissue, cell, subject, etc.), whereas in otherembodiments, the cDNAs that are sequenced may be a pool of nucleic acidsextracted from a plurality of sources (e.g., a pool of nucleic acidsfrom a plurality of organisms, tissues, cells, subjects, etc.), where by“plurality” is meant two or more. As such, in certain embodiments, cDNAsthat are sequenced can contain nucleic acids from 2 or more sources, 3or more sources, 5 or more sources, 10 or more sources, 50 or moresources, 100 or more sources, 500 or more sources, 1000 or more sources,5000 or more sources, up to and including about 10,000 or more sources.Molecular barcodes may allow the sequences from different sources to bedistinguished after they are analyzed. The sequence reads may beanalyzed by a computer and, as such, instructions for performing thesteps set forth below may be set forth as programming that may berecorded in a suitable physical computer readable storage medium.

The method described herein can be employed to analyze mRNA and,optionally, small RNAs from virtually any organism and/or sample-type,including, but not limited to, plants, animals (e.g., reptiles, mammals,insects, worms, fish, etc.), tissue samples, cadaveric tissue,archaeological/ancient samples, etc. In certain embodiments, the RNAsample used in the method may be derived from a mammal, where in certainembodiments the mammal is a human. In exemplary embodiments, the RNAsample may contain RNA from a mammalian cell, such as, a human, mouse,rat, or monkey cell. The sample may be made from cultured cells or cellsof a clinical sample, e.g., a tissue biopsy, scrape or lavage or cellsof a forensic sample (i.e., cells of a sample collected at a crimescene). In particular embodiments, the RNA sample may be obtained from abiological sample such as cells, tissues, bodily fluids, and stool.Bodily fluids of interest include but are not limited to, blood, serum,plasma, saliva, mucous, phlegm, cerebral spinal fluid, pleural fluid,tears, lactal duct fluid, lymph, sputum, cerebrospinal fluid, synovialfluid, urine, amniotic fluid, and semen. In particular embodiments, asample may be obtained from a subject, e.g., a human. In someembodiments, the sample analyzed may be a sample of cfRNA obtained fromblood, e.g., from the blood of a pregnant female or a patient.

The present method may be employed in a variety of diagnostic, drugdiscovery, and research applications that include, but are not limitedto, diagnosis or monitoring of a disease or condition (where theexpression of an mRNA and/or small RNA provides a marker for the diseaseor condition), discovery of drug targets (where an mRNA and/or small RNAis differentially expressed in a disease or condition and may betargeted for drug therapy), drug screening (where the effects of a drugare monitored by assessing the level of an mRNA and/or small RNA),determining drug susceptibility (where drug susceptibility is associatedwith a particular profile of an mRNA and/or small RNA) and basicresearch (where is it desirable to identify the presence of an mRNAand/or small RNA in a sample, or, in certain embodiments, the relativelevels of a particular mRNA and/or small RNA in two or more samples).

In certain embodiments, relative levels of an mRNA and/or small RNA intwo or more different small RNA samples may be obtained using the abovemethods, and compared. In these embodiments, the results obtained fromthe above-described methods are usually normalized to the total amountof RNA in the sample or to control RNAs (e.g., constitutive RNAs), andcompared. This may be done by comparing ratios, or by any other means.In particular embodiments, the mRNA and/or small RNA profiles of two ormore different samples may be compared to identify mRNAs and/or smallRNAs that are associated with a particular disease or condition (e.g.,an mRNA and/or small RNA that is induced by the disease or condition andtherefore may be part of a signal transduction pathway implicated inthat disease or condition).

The different samples may consist of an “experimental” sample, i.e., asample of interest, and a “control” sample to which the experimentalsample may be compared. In many embodiments, the different samples arepairs of cell types or fractions thereof, one cell type being a celltype of interest, e.g., an abnormal cell, and the other a control, e.g.,a normal cell. If two fractions of cells are compared, the fractions areusually the same fraction from each of the two cells. In certainembodiments, however, two fractions of the same cell may be compared.Exemplary cell type pairs include, for example, cells isolated from atissue biopsy (e.g., from a tissue having a disease such as colon,breast, prostate, lung, skin cancer, or infected with a pathogen etc.)and normal cells from the same tissue, usually from the same patient;cells grown in tissue culture that are immortal (e.g., cells with aproliferative mutation or an immortalizing transgene), infected with apathogen, or treated (e.g., with environmental or chemical agents suchas peptides, hormones, altered temperature, growth condition, physicalstress, cellular transformation, etc.), and a normal cell (e.g., a cellthat is otherwise identical to the experimental cell except that it isnot immortal, infected, or treated, etc.); a cell isolated from a mammalwith a cancer, a disease, a geriatric mammal, or a mammal exposed to acondition, and a cell from a mammal of the same species, preferably fromthe same family, that is healthy or young; and differentiated cells andnon-differentiated cells from the same mammal (e.g., one cell being theprogenitor of the other in a mammal, for example). In one embodiment,cells of different types, e.g., neuronal and non-neuronal cells, orcells of different status (e.g., before and after a stimulus on thecells) may be employed. In another embodiment of the invention, theexperimental material is cells susceptible to infection by a pathogensuch as a virus, e.g., human immunodeficiency virus (HIV), etc., and thecontrol material is cells resistant to infection by the pathogen. Inanother embodiment of the invention, the sample pair is represented byundifferentiated cells, e.g., stem cells, and differentiated cells.

In some embodiments, the sequence reads may be analyzed to provide aquantitative determination of which sequences are in the sample. Thismay be done by, e.g., counting sequence reads or, alternatively,counting the number of original starting molecules, prior toamplification, based on their fragmentation breakpoint and/or whetherthey contain the same indexer sequence (which can be present in the 5′adaptor, for example). The use of molecular barcodes in conjunction withother features of the fragments (e.g., the end sequences of thefragments, which define the breakpoints) to distinguish between thefragments is known. Molecular barcodes and exemplary methods forcounting individual molecules are described in Casbon (Nucl. Acids Res.2011, 22 e81) and Fu et al (Proc Natl Acad Sci USA. 2011 108: 9026-31),among others. Molecular barcodes are described in US 2015/0044687, US2015/0024950, US 2014/0227705, U.S. Pat. Nos. 8,835,358 and 7,537,897,as well as a variety of other publications.

Also provided is a method for identifying a pattern that correlates withphenotype, e.g., a disease, condition or clinical outcome, etc. In someembodiments, this method may comprise (a) performing the above-describedmethod on a plurality of RNA samples, wherein the RNA samples areisolated from patients having a known phenotype, e.g., disease,condition or clinical outcome, thereby determining which RNAs from eachof the patients; and (b) identifying a signature that is correlated withthe phenotype.

In some embodiments, the signature may be diagnostic (e.g., may providea diagnosis of a disease or condition or the type or stage of a diseaseor condition, etc.), prognostic (e.g., indicating a clinical outcome,e.g., survival or death within a time frame) or theranostic (e.g.,indicating which treatment would be the most effective).

Also provided is a method for analyzing a patient sample. In thisembodiment, the method may comprise: (a) identifying, using theabove-described method, sequences that are under and/or over expressedin a patient; (b) comparing the identified sequences to a set ofsignature sequences that are correlated with a phenotype, e.g., adisease, condition, or clinical outcome etc.; and (c) providing a reportindication a correlation with phenotype. This embodiment may furthercomprise making a diagnosis, prognosis or theranosis based on theresults of the comparison.

In some embodiments, the method may involve creating a report (anelectronic form of which may have been forwarded from a remote location)and forwarding the report to a doctor or other medical professional todetermine whether a patient has a phenotype (e.g., cancer, etc.) or toidentify a suitable therapy for the patient. The report may be used as adiagnostic to determine whether the subject has a disease or condition,e.g., a cancer. In certain embodiments, the method may be used todetermine the stage or type cancer, to identify metastasized cells, orto monitor a patient's response to a treatment, for example.

In any embodiment, a report can be forwarded to a “remote location”,where “remote location,” means a location other than the location atwhich the image is examined. For example, a remote location could beanother location (e.g., office, lab, etc.) in the same city, anotherlocation in a different city, another location in a different state,another location in a different country, etc. As such, when one item isindicated as being “remote” from another, what is meant is that the twoitems can be in the same room but separated, or at least in differentrooms or different buildings, and can be at least one mile, ten miles,or at least one hundred miles apart. “Communicating” informationreferences transmitting the data representing that information aselectrical signals over a suitable communication channel (e.g., aprivate or public network). “Forwarding” an item refers to any means ofgetting that item from one location to the next, whether by physicallytransporting that item or otherwise (where that is possible) andincludes, at least in the case of data, physically transporting a mediumcarrying the data or communicating the data. Examples of communicatingmedia include radio or infra-red transmission channels as well as anetwork connection to another computer or networked device, and theinternet or including email transmissions and information recorded onwebsites and the like. In certain embodiments, the report may beanalyzed by an MD or other qualified medical professional, and a reportbased on the results of the analysis of the image may be forwarded tothe patient from which the sample was obtained.

Accordingly, among other things, the instant methods may be used to linkthe expression of certain genes to certain physiological events.

EMBODIMENTS Embodiment 1

A method for processing an RNA sample, comprising,

(a) fragmenting a sample comprising RNA to produce a population of RNAfragments of different lengths, wherein the population of RNA fragmentscomprises longer RNA fragments and shorter RNA fragments,

(b) incubating the population of RNA fragments with a polyA polymeraseunder conditions wherein the polyA polymerase preferentially adds apolyA tail to the longer RNA fragments and not the shorter fragments, toproduce tailed RNA;

(c) hybridizing the tailed RNA to an oligo(dT) oligonucleotide; and

(d) either (i) reverse transcribing the tailed RNA using the oligo(dT)oligonucleotide as a primer, to produce a cDNA library or (ii) enrichingfor the tailed RNA by washing away RNA molecules that are not hybridizedto the oligo(dT) oligonucleotide.

Embodiment 2

The method of claim 1, wherein the RNA sample comprises at least mRNA.

Embodiment 3

The method of any prior claim, wherein the longer fragments are at least20 ribonucleotides in length and the shorter fragments up to 15ribonucleotides in length.

Embodiment 4

The method of any prior claim, wherein the conditions of step (b)comprise 0.6 mM to 1.2 mM of a divalent cation.

Embodiment 5

The method of any prior claim, wherein the conditions of step (b)comprise 0.75 mM to 1.0 mM of a divalent cation.

Embodiment 6

The method of claim 4 or 5, wherein the divalent cation is Mg²⁺.

Embodiment 7

The method of any of claims 1-3, wherein the conditions of step (b)comprise polyA polymerase at a concentration of less than 0.02 U/uL.

Embodiment 8

The method of any of claims 1-3, wherein the conditions of step (b)comprise polyA polymerase at a concentration in the range of 0.002 U/uLto 0.02 U/uL.

Embodiment 9

The method of any prior claim, wherein the sample of step (a) comprisesDNA:RNA hybrids, and the method comprises treating the sample withRNAseH.

Embodiment 10

The method of any of claims 1-8, wherein step (a) is done by heating theRNA in the presence of a divalent cation.

Embodiment 11A

The method of any prior claim, wherein the sample of step (a) furthercomprises small RNAs, wherein: the population of RNA fragments of step(a) comprises: i. longer RNAs comprising unfragmented small RNAs andfragments of the RNA and ii, shorter RNAs comprising fragments of theRNA, the tailed RNA of step (b) contains tailed RNA fragments and tailedunfragmented small RNAs.

Embodiment 11B

The method of any prior claim, wherein the sample of step (a) furthercomprises small RNAs, wherein the method comprises:

(a) fragmenting the sample to produce a population of RNA fragments ofdifferent lengths, wherein the population of RNA fragments comprises: i.longer RNAs comprising unfragmented small RNAs and fragments of the RNAand ii, shorter RNAs comprising fragments of the RNA,

(b) incubating the population of RNA fragments with a polyA polymeraseunder conditions wherein the polyA polymerase preferentially adds apolyA tail to the longer RNAs and not the shorter RNAs, to producetailed RNA that contains tailed RNA fragments and tailed unfragmentedsmall RNAs; and

(c) hybridizing the tailed RNA to an oligo(dT) oligonucleotide; and

(d) reverse transcribing the tailed RNA using the oligo(dT)oligonucleotide as a primer, to produce a cDNA library or enriching forthe tailed RNA by washing away RNA molecules that are not hybridized tothe oligo(dT) oligonucleotide.

Embodiment 12

The method of claim 11, wherein the small RNAs include microRNA (miRNA)molecules, tiny non-coding RNA (tncRNA) molecules, small modulatory RNA(smRNA) molecules, Piwi-interacting RNA (pRNA) molecules, and snoRNAmolecules.

Embodiment 13

The method of any prior claim, further comprising sequencing the cDNAlibrary or the enriched tailed RNA.

Embodiment 14

The method of claim 13, wherein the method comprises amplifying the cDNAlibrary by PCR, prior to sequencing.

Embodiment 15

The method of any prior claim, wherein the method further comprisesadding an adaptor sequence to the 5′ end of the population of RNAfragments of (a) or the tailed RNA of (b).

EXAMPLES

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Example 1

Except for changes to the polyadenylation step, a total of 20 ng MCF7total RNA was fragmented, polyadenylated, ligated to adaptors, reversetranscribed and amplified essentially as described in U.S. patentapplication Ser. No. 15/818,469, filed on Nov. 20, 2017, andincorporated by reference herein. Libraries were sequenced on anIllumina MiSeq. Fastq files were generated and reads were trimmed usingcutadapt then aligned to a human transcriptome reference. Samtools wasused to count mRNA-aligned reads at each insert length.

In the first experiment, different amounts of poly(A) polymerase wereadded to the polyadenylation reaction. In this experiment, thepolyadenylation reaction of condition A (15 uL) contains 1 uL ofundiluted poly(A) polymerase (5 U/uL) and 10 mM MgCl₂ in a 1× buffer,whereas the polyadenylation reaction in condition B (15 ul) contains 1ul of a 1/48 dilution of poly(A) polymerase (5 U/uL, diluted in itsenzyme storage buffer) and 10 mM MgCl₂ in a 1× buffer. The finalconcentration of the enzyme in condition A is approximately 0.33 U/uLwhereas the final concentration of the enzyme in condition B isapproximately 0.007 U/uL. In total, condition A uses 5 U of the enzymeand condition B uses approximately 0.10 U of the enzyme.

The results of the first experiment are shown in FIG. 4. These resultsshow that that there is much less bias towards shorter sequence reads(i.e., reads for shorter fragments) if a 1/48 dilution of the poly(A)polymerase is used.

In the second experiment, the polyadenylation reaction was done usingdifferent amounts of MgCl₂. MgCl₂ is required for poly(A) polymerase andreducing the amount of MgCl₂ decreases the activity of the enzyme. Inthis experiment, the polyadenylation reaction in condition C contains 1ul of undiluted poly(A) polymerase and 10 mM MgCl₂ in a 1× reaction,whereas the polyadenylation reaction in condition D contains 1 ul ofundiluted poly(A) polymerase and 1 mM MgCl₂ in a 1× reaction.

The results of the second experiment are shown in FIG. 5. These resultsshow that that there is much less bias towards shorter sequence reads(i.e., reads for shorter fragments) if the polyadenylation reaction isdone in a reaction buffer that contains 1 mM MgCl₂.

That which is claimed is:
 1. A reagent system for making cDNA fromfragmented RNA comprising: (a) a first oligod(T) primer; (b) a reversetranscriptase; (c) a thermostable RNaseH; (d) a poly(A) polymerase; (e)a 5′ adapter; (f) an RNA ligase; and (g) a second oligod(T) primer,wherein the second oligod(T) primer comprises a 5′ tail.
 2. The systemof claim 1, wherein the RNaseH is active at a temperature in the rangeof 60° C. to 80° C.
 3. The system of claim 1, wherein the firstoligod(T) primer is an anchored oligo(dT) primer.
 4. The system of claim1, wherein the first oligod(T) primer does not have a 5′ tail.
 5. Thesystem of claim 1, wherein the system further comprises: (h) a pair ofPCR primers, wherein the pair of PCR primers comprises a first primerthat hybridizes to the 5′ tail of the second oligod(T) primer and asecond primer that hybridizes to the complement of the 5′ adapter.